Question

A protein sequence in solution at temperature T folds from a denatured extended state to its native state. Under which of the following conditions on changes in enthalpy (H) and entropy (S) should such a transition be NOT favorable?
A) 0 > H > TS
B) H < 0 < TS
C) 0 > TS > H
D) H = 0 and TS > 0​

Answer 1

Answer:

It remains true that chemical denaturants, guanidine hydrochloride and urea, are the most generally applicable methods of completely unfolding proteins. Both of these molecules have a low molecular weight and are extremely soluble, such that 6–8 molar concentrations will denature virtually any protein. Computational studies of polypeptides interacting with these molecules have revealed some aspects of their denaturing power 10, although far more is understood about urea. The molecular dynamics of an unfolded protein indicate that urea readily forms hydrogen bonds with the peptide backbone, disrupts native contacts, and makes extended conformations favorable 11. Simulations comparing urea and guanidine on the same protein find that guanidine does not make many hydrogen bonds but does disrupt hydrophobic interactions within the native state, particularly between aromatic side chains 12.

A number of studies of various proteins in high denaturant have shown that these chains are acting as self-avoiding random polymers 13, 14. Measurement of intramolecular contact of unstructured peptides in water with guanidine and water with urea showed that a wormlike chain with excluded volume is a better model than a freely jointed chain, but the persistence length (4–6 Å) and excluded diameter (4 Å) are sufficiently small that a freely jointed chain is a good model for proteins of any reasonable length 15, 16. The intramolecular diffusion coefficients measured by these same experiments reveal values in the 10 -6 cm 2/s range for all sequences in high denaturant, about the same as the translational diffusion coefficient for objects of this size 17– 20. Thus, the view of unfolded proteins as completely random, freely diffusing polymers appears to be justified in high denaturant.

The difficulty with using denaturant as an unfolding mechanism is the technical challenges with rapidly diluting it to prompt refolding in a kinetic experiment. The dilution or mixing time is a period in which the solution conditions are not in equilibrium and folding kinetics are not in response to a known set of conditions. Conventional stopped-flow mixers have “dead times”, the time during which measurement is not possible, of 1–5 ms that are determined by the turbulence induced in the mixing process, yet folding may still be occurring. Smaller turbulent mixers have pushed this dead time down to as low as 30 μs 21, 22. Laminar flow mixers developed in my lab have eliminated turbulence and mix as fast as 2–4 μs 23– 26.